Our long term objective is to develop molecules capable of specifically recognizing and modifying defined sequences of DNA. These studies are expected to provide increased insight into the molecular basis for selective recognition of nucleic acids by proteins, oligonucleotides and small molecules. In addition, this work may result in new tools for manipulating and analyzing nucleic acid structure and function, as well as therapeutic agents based on selective gene inactivation. During the period of the previous application we addressed the problem of sequence specific DNA modification, specifically the design of molecules that hydrolyze nucleic acids at predefined sequences. A combination of chemical and biological mutagenesis was used to convert the relatively nonspecific phosphodiesterase, staphylococcal nuclease, into a molecule capable of sequence specifically hydrolyzing RNA, single-stranded DNA and duplex DNA. Adducts of staphylococcal nuclease with either oligonucleotides or DNA-binding proteins selectively bound and hydrolyzed RNA and DNA via Watson-Crick base pairing interactions, D-loop formation, triple-helix formation, or specific protein DNA complex formation. A major limitation to further progress in the development of these "engineered nucleases" is the availability of molecules capable of recognizing any predefined sequence of duplex DNA. Consequently, the focus of this continuation is (a) to increase our understanding of the molecular basis for the selective recognition of DNA by polypeptides and oligonucleotides and (b) to develop new strategies for generating oligonucleotides and proteins that selectively bind any predefined sequence of double-stranded DNA. This will be accomplished via a combination of genetic and chemical approaches. Large libraries (greater than 10-8) of proteins and oligonucleotides will be generated and screened (by in vitro affinity chromatography or in vivo selections) in order to identify "ligands" that bind specific target sequences. The resulting ligands will be characterized with respect to their affinity and specificity for DNA. The insight gained from this combinatorial approach to DNA recognition will be complemented by a detailed analysis of the interactions between DNA and sequence specific DNA binding proteins. Amino acids in the structurally characterized DNA binding proteins, 434 repressor and CAP, will be substituted with a series of unnatural amino acid variants with precisely defined electronic and steric properties. Characterization of these mutant repressors should allow us to more precisely define the structural features responsible for the selective recognition of DNA by proteins.